PROJECT SUMMARY/ABSTRACT This proposal represents the renewal of a grant that was converted to an R37 MERIT award in 2007. Its long- term objective has been centered on identifying key cytoskeletal components that orchestrate membrane and receptor trafficking pathways critical to liver physiology. Recently, we have expanded our efforts to additionally examine the contributions of autophagy, due to its direct relevance in hepatic function and the important fact that this process shares significant mechanistic overlap with the secretory and endocytic pathways. Non- alcoholic fatty liver disease (NAFLD), with underlying hepatic steatosis and inflammation, is rapidly becoming a prominent health issue. This proposal is focused on the fundamental mechanisms that regulate the autophagic hepatocellular breakdown and utilization of unique triglyceride-rich organelles, lipid droplets (LDs), for use as an energy source by mitochondria. Significant evidence implicates the selective targeting and breakdown of hepatic LDs by the autophagic machinery in a process called lipophagy. We, and others, have demonstrated that important components of this essential cellular process are large and small guanosine triphosphatases (GTPases). These include the Ras-like Rab GTPases that control many membrane-trafficking processes in the hepatocyte and the Dynamin (Dyn) family of large GTPase mechanoenzymes that mediate membrane scission/fusion throughout the cell. We have made seminal contributions toward the identification and functional analysis of these enzymes in the hepatocyte. We have recently found that they play an important role in hepatic lipophagy; specifically, that a subset of Rab GTPases mediate the interaction, docking, and envelopment of LD in the hepatocyte by nascent autophagic membranes. We have also observed that a subset of these Rabs appear to bind Dyn proteins at the LD and mitochondria to regulate lipid metabolism. These findings support this proposal's central hypothesis that large and small GTPases work synergistically to mediate lipophagy and catabolism in the hepatocyte by regulating the interactions between LDs, autophagic membranes, and mitochondria. The following specific aims represent novel and innovative biological concepts in this proposal: 1) determining how a specific sub-family of Rab GTPases (Rabs 8, 10, and 13) interact with the large GTPase Dyn2 to coordinate the establishment and extension of the phagophore during hepatic lipophagy; 2) defining the mechanism by which the autophagic phagophore extends around the LD during lipophagy in the hepatocyte through the action of a novel membrane-deforming Rab effector protein complex (EHD2 and EHBP1); and 3) defining the role of mitochondrial-associated Dyns in the structural relationship between the LD, autolysosome, and mitochondria during hepatic energy production. Completion of these studies will provide valuable insights into hepatocellular lipid metabolism, resulting in a better understanding of the underlying basis for hepatic steatosis, leading to novel strategies for therapeutic intervention in NAFLD.